The invention relates generally to methods of screening complex biological materials for effective regulatory, therapeutic, or diagnostic compounds. The invention encompasses using capillary electrophoresis and mass spectrometry together in a method particularly advantageous for detecting and characterizing tight-binding ligands in mixtures that may also include much higher concentrations of competing, weaker-binding ligands. The method also allows ranking of ligands according to their relative binding strengths.
Developing screening protocols to identify new, biologically active compounds can present unique and difficult challenges, especially when screening complex materials, particularly a xe2x80x9ccomplex biological samplexe2x80x9d (CBS): any sample of material that may have an effect in a biological system. Examples of CBS include but are not limited to: a natural product; a natural extract; a biological preparation; a chemical mixture; a pure compound library; and a combinatorial library.
While capillary electrophoresis has previously been used to detect and/or to analyze known compounds and materials of known composition, this technology has not been widely used, until recently, to screen complex biological samples for target-binding compounds that were previously unknown or unidentified as being ligands to a selected target molecule.
For example, WO 97/22000 encompasses four broad embodiments of a capillary electrophoretic screening method for unknown, biologically active compounds, as follows.
(1) In a non-competitive embodiment of WO 97/22000, a target and complex biological sample are mixed together, then an aliquot of that target/sample mixture is subjected to capillary electrophoresis (CE), and the CE migration of the target is tracked. The target""s migration pattern under these conditions are compared against a reference standard, typically the unbound target""s migration pattern in the absence of any target-binding ligand.
(2) In a non-competitive, subtractive analysis embodiment of WO 97/22000, a target and sample are mixed together and then subjected to CE. The migration pattern of this mixture is compared to the migration pattern of a sample of the complex biological material alone. Any difference between the two migration patterns suggests the presence in the sample of a hit compound that can bind to the target.
(3) One competitive binding embodiment is provided in WO 97/22000, which tracks known, charged ligand: The target is first mixed with a complex biological material sample and then with a known, charged ligand that binds tightly to the target, to form a sample/target/known ligand mixture. This method uses an essentially equilibrium setting when incubating target and known, tight-binding ligand together, so that the known, tight-binding ligand can displace any weaker-binding hit, prior to CE. This mixture is subjected to capillary electrophoresis and the migration of the known, charged ligand is tracked. (Thus, this method is useful when the target is not easily detected during CE.) Any difference in the known, charged ligand""s migration pattern, when in the presence of both the target and a complex biological material sample, from the known ligand""s migration pattern when in the presence of the target alone, indicates the presence of a candidate, unidentified target-binding ligand in that sample.
(4) In another competitive binding embodiment of WO 97/22000, the target""s migration is tracked and the CE running buffer contains a known, weak-binding, competitive ligand. The target is mixed with a sample, and an aliquot of the mixture is subjected to CE in the presence of a known, relatively weak, target-binding xe2x80x98competitorxe2x80x99 ligand in the CE running buffer. The migration of the target is tracked during CE. The reference standard is the migration of a target plug alone in the known ligand-containing CE buffer, its migration being shifted by its weak, reversible binding to the known ligand dispersed in the CE buffer, as compared to the target""s migration alone ligand-free buffer. This competitive screening method can detect a tight-binding hit compound in a target/natural sample mixture, because the hit binds up the target for the entire CE run and prevents the target""s interaction with the known weak-binding ligand in the buffer. Therefore, the CE migration pattern of the target in the sample/target aliquot would shift back to the target""s migration position as it would be in ligand-free running buffer. This method, too, is particularly useful when the unbound target is not easily detected in ligand-free buffer during CE.
While WO 97/22000 provides useful CE screening methods, they do not overcome some common drug-screening problems. A major obstacle to successful and cost-effective drug screening has been the presence of high concentrations of one or several weak, target-binding ligand compound(s) in a screened sample, which can mask the presence of more valuable, moderate-to-tight-binding or tight-binding ligands occurring at a lower concentration within the same sample. Another major obstacle is obtaining structural information about the high affinity ligands, especially when they are present in very complex mixtures.
Therefore, there remains a need for rapid and cost-effective screening tools for discovering new bioactive and/or potential regulatory compounds that bind to molecules involved in disease or essential molecules of key metabolic pathways. Also needed is a way of characterizing those candidate ligands displaying the highest binding strengths to the target.
The present invention answers these needs by providing an improved screening method combining both capillary electrophoresis and mass spectrometry techniques. Capillary electrophoresis, specifically capillary zone electrophoresis, enables rapid and cost-effective separation and identification of compounds in a sample while consuming only minute amounts of the sample. In the particular application taught here, capillary electrophoresis (CE) enables selective identification of particular candidate ligand(s) that bind(s) tightly to a target of interest. The CE steps are optimized to screen out all but those ligands that bind to the target molecule of interest at or above a selected binding strength, as taught in International Application No. PCT/US98/27463, herein incorporated by reference.
Mass spectrometry (MS) enables analysis of biomolecules, such as peptides and proteins, at the molecular level with high mass measurement accuracy. Suitable MS ionization techniques include, but are not limited to electron impact ionization (EI), electrospray ionization (ESI), chemical ionization (CI), atmospheric-pressure chemical ionization (APCI), matrix-assisted-laser-desorption ionization (MALDI), thermospray (TSP), and fast atom bombardment (FAB) ionization. These ionization techniques may be combined with time-of-flight (TOF), single or triple quadrupole, Fourier transform, or ion trap MS analysis to provide additional information of the compounds analyzed. For example, MALDI-TOF mass spectrometry is valued for ease of sample preparation, predominance of singly charged ions in mass spectra, sensitivity, and high speed. Ion trap and Fourier transform MS allow re-analysis of the ions, as needed. If desired, the ions may be subjected to fragmentation, such as collision-induced dissociation (CID), during mass spectrometry to provide additional structural or substructural data.
Moreover, since MS allows compounds to be detected and differentiated by their molecular weight and/or size, it may be used to observe selectively the target""s migration pattern after undergoing CE either alone or in the presence of a complex biological sample. Therefore, MS allows one to eliminate any separate detector and/or any derivatization of the target, if one so desires.
Alternatively, one may use a separate detector, e.g. an ultraviolet absorbance (UV) or fluorescence detector (e.g., light-induced fluorescence (LIF), at a point along the CE capillary or microchip, to track the target""s migration during CE, and to generate a CE profile separate from mass spectrometry data.
Identifying and immediately characterizing those candidate ligands that form the most stable complexes with the selected target, minimizes the time and resources needed for isolating and characterizing these compounds. The invention provides a more cost-effective screening protocol since the most stable target-binding ligands are those most likely to be effective therapeutic, regulatory, and/or diagnostic compounds and drugs.
The method generally combines a capillary electrophoresis (CE) technique for screening complex biological samples with a mass spectrometry (MS) analysis step to provide a streamlined procedure for selectively identifying and characterizing any candidate ligand(s) in a complex biological sample that binds at a selected binding strength to a selected target molecule. Advantageously, the CE/MS method can selectively identify and characterizes moderate-to-tight binding ligands (MTLs), especially tight-binding ligands (TLs), even in the presence of high concentrations of weak ligands (WLs) that often mask lower concentrations of tight-binding ligands in the same sample. As a result, the present method improves over prior CE screening methods, by selectively detecting TLs over WLs, and by providing valuable structural data about candidate tight-binding ligands, all in one procedure. Alternatively, as desired, one can adjust the CE and other conditions of the method so that weaker binding ligands are detected. The present method also allows the ranking of target-binding ligands detected in a complex biological sample according to their relative binding strengths.
Mass spectrometric analysis of compounds screened and separated by capillary electrophoresis offers the advantages of rapidly obtaining structural characterization of high-affinity ligands identified by CE. As well, the mass spectrometer itself can be used to track the CE migration of the target, by selectively monitoring the mass/net charge (m/z) ratio of the ionized target, and thus to selectively detect any candidate ligand of a desired binding strength. Using the mass spectrometer as a detector eliminates the need to use any additional detector and the need to label or otherwise derivatize the target to make it detectable during CE.
Specifically, the method involves interfacing a capillary or microfabricated chip of a CE instrument with a post-capillary mass spectrometer. A suitable interface is provided by, e.g, the microscale fluid handling system disclosed in Karger et al., U.S. Pat. No. 5,872,010. The present method provides direct mass and structural analysis of candidate ligands that form target/ligand complexes that migrate stably through the CE instrument, a detectable amount (e.g., at least 50% or at least 80%) of which remains tightly bound, i.e., for a substantial portion of the CE run time, preferably at least 50% or, even better, at least 80%, most preferably until they reach the outlet end of the CE capillary. All weaker-binding ligands will remain undetected during the MS analysis because they substantially dissociate from the target before reaching the outlet end of the capillary and entering the mass spectrometer, preferably early during the CE run. Therefore, the present method can preferentially identify and structurally characterize tight-binding ligands in complex biological samples.
In summary, the present method screens complex biological material for and characterizes any candidate ligand that binds to a selected target at or above a selected binding strength, by the steps disclosed herein.
In one embodiment, a complex biological sample is combined with a selected target to form a sample/target mixture. A plug of the sample/target mixture is then injected into an inlet end of a conduit of a capillary electrophoresis instrument (e.g., a capillary or a channel of a microchip). The compounds within the sample/target plug are subjected to capillary electrophoresis (CE) under predetermined conditions. The predetermined CE conditions have been optimized so that any first complex, formed between the target and any candidate ligand binding to the target at or above a selected binding strength (e.g., a tight binder), remains bound for a substantial part the capillary electrophoresis run time, and so that any additional complex(es), formed between the target and any additional ligand(s) binding to the target below the selected binding strength (e.g. a weak binder), dissociates prior to reaching the CE/MS interface at the outlet end of the CE conduit. In at least one embodiment, the migration of the target during capillary electrophoresis is tracked, enabling one to obtain at least one capillary electrophoretic profile of the sample/target plug. The target""s migration may be tracked by, e.g., an ultraviolet light absorbance or fluorescence detector coupled to the CE capillary. Alternatively, on-line MS detection can be used exclusively, obviating the need for absorbance or fluorescence detection. The compounds from the electrophoresed sample/target plug are introduced from the capillary electrophoresis instrument into an on-line mass spectrometer interfaced with the CE instrument. The compounds from the electrophoresed sample/target plug are subjected to ionization and then mass spectrometry analysis (i.e., the mass spectra of the ions are analyzed). The mass spectrometry data are gathered.
One obtains and analyzes the CE profile of the sample/target and target plugs, i.e., the migration pattern of the unbound or bound target upon capillary electrophoresis. One may look at either a time shift in the target""s migration peak, resulting from it being bound by a moderate-to-tight-binding ligand. Alternatively, particularly if using fluorescence or absorbance detection, or single-ion monitoring during MS, one can look at other parameters, such as the shape, area, or size of the unbound target peak and/or any bound target/ligand complex peak(s) detected, and any change(s) in at least one of those parameters.
One analyzes at least one capillary electrophoretic profile generated from the present CE/MS screening method (from the absorbance, fluorescence, or MS data), by comparing it to at least one reference standard. From that comparison, one determines whether the capillary electrophoretic profile indicates the presence of a candidate target-binding ligand, typically but not necessarily an unidentified one, in the complex biological sample. As well, the mass spectrometry data of the ionized sample/target compounds are analyzed to determine the mass and other structural data of any detected target-binding ligand, including a candidate ligand having the desired binding strength. Structural data may be provided by performing collision-induced dissociation (CID) mass spectrometry or other appropriate fragmentation process, e.g., post-source decay, in-source fragmentation, etc.